Lung cancer organoid and peripheral blood source immune cell co-culture model and construction method thereof

Abstract

The invention relates to a lung cancer organoid and peripheral blood source immune cell co-culture model and a construction method thereof. Specifically, the invention provides a construction method of a macrophage and tumor organoid co-culture model for evaluating anti-tumor activity, monocytes of autologous or allogeneic peripheral blood of a patient are induced and differentiated into high-purity macrophages in vitro, and the high-purity macrophages and lung cancer tumor organoid are subjected to three-dimensional co-culture in matrigel; the model aims at highly reducing a core interaction network of tumor cells and macrophages in TME; the dynamic change of the polarization state of the macrophage is simulated and observed; the problem that an organ-like model is incomplete due to immune component deficiency or spatial positioning distortion of an existing model is effectively solved, an experimental platform closer to the physiological state is provided for tumor immune microenvironment research, and therefore development of the tumor immune treatment field is promoted.

Description

Technical Field

[0001] This invention relates to the field of tumor organoid model construction technology, and more specifically, to a lung cancer organoid co-culture model with peripheral blood-derived immune cells and its construction method. Background Technology

[0002] Lung cancer is a major disease that seriously threatens human health. Reliable research models are crucial for advancing its treatment, but existing models have significant limitations. Two-dimensional cell culture lacks three-dimensional spatial structure and microenvironment, making it difficult to realistically simulate the biological behavior of tumors in vivo; animal models have species differences, and their immune systems and physiological metabolism differ from humans, making it difficult to accurately reflect the characteristics and immune responses of human tumors; while patient-derived xenograft models are closer to the actual situation of human tumors, they are difficult to model, have long cycles, low success rates, and cannot achieve high-throughput drug screening, thus limiting their clinical translational applications.

[0003] With the rise of organoid technology, patient-derived tumor organoids (PDOs) have become an important tool for next-generation cancer research due to their unique advantages, such as high reproducibility of tumor tissue heterogeneity, three-dimensional spatial structure, and genomic characteristics. However, existing PDO models mainly focus on tumor cells themselves, and the reconstruction of the tumor immune microenvironment (TME) remains insufficient. Of particular note is that existing immune co-culture studies have largely focused on peripheral blood or tumor tissue-derived T lymphocytes, neglecting the crucial component of tumor-associated macrophages (TAMs). In fact, TAMs account for 30%-50% of tumor immune cells and play a central role in lung cancer progression, metastasis, and immunotherapy resistance by secreting immunosuppressive cytokines (such as TGF-β and IL-10), remodeling the extracellular matrix (ECM), and regulating angiogenesis. More importantly, existing models not only lack effective integration of TAMs but also lack the ability to simulate the dynamic regulation of their polarization state (M1 / M2 type). This significantly limits the potential for in-depth analysis of the complex molecular mechanisms of the "tumor-immune" interaction, especially in exploring therapeutic strategies targeting TAMs.

[0004] Therefore, there is an urgent need in this field to develop a simple, high-fidelity three-dimensional co-culture biomimetic model system for lung cancer tumor organoids and macrophages, and its construction method. The aim is to highly replicate the core interaction network between tumor cells and macrophages (especially TAMs) in the tumor immune microenvironment (TME); and to simulate and observe the dynamic changes in macrophage polarization state; effectively addressing the problems of incomplete organoid models caused by the lack of immune components or spatial localization distortion in existing models, providing a more physiologically accurate experimental platform for tumor immune microenvironment research, thereby promoting the development of the field of tumor immunotherapy. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide an innovative three-dimensional co-culture biomimetic model system and its construction method. The core technical solution involves: in vitro induction of high-purity macrophages from autologous or allogeneic peripheral blood mononuclear cells, followed by three-dimensional co-culture with lung cancer tumor organoids in a matrix gel. This model aims to highly replicate the core interaction network between tumor cells and macrophages (especially TAMs) in the tumor immune microenvironment (TME); and to simulate and observe the dynamic changes in macrophage polarization. It effectively solves the problems of incomplete organoid models caused by missing immune components or distorted spatial positioning in existing models, providing a more physiologically-like experimental platform for tumor immune microenvironment research, thereby promoting the development of the field of tumor immunotherapy.

[0006] In a first aspect of the present invention, a method for constructing a macrophage and tumor organoid co-culture model for evaluating antitumor activity is provided, comprising the following steps:

[0007] (1) Peripheral blood mononuclear cells were isolated from peripheral blood of healthy individuals, and macrophage cells were induced to be obtained after macrophage pretreatment culture and induction culture.

[0008] (2) The tumor tissue from the cancer patient was digested with digestive fluid, the digested tumor cells were collected by centrifugation and mixed evenly with matrix gel, and then added to tumor organoid culture medium for tumor organoid culture and passage culture to obtain humanized tumor organoids.

[0009] (3) The humanized tumor organoids obtained in step (2) and the macrophages obtained in step (1) are mixed at an effector-target ratio of 1 to 10:1 to form a co-culture system, and then placed in an incubator for culture to obtain the macrophage and tumor organoid co-culture model used to evaluate anti-tumor activity.

[0010] In another preferred embodiment, the tumor is a malignant tumor.

[0011] In another preferred embodiment, the tumor is lung cancer.

[0012] In another preferred embodiment, the culture medium for macrophage pretreatment culture in step (1) includes: DMEM high glucose medium, 10% fetal bovine serum, 1% GlutaMAX and 1% penicillin-streptomycin.

[0013] In another preferred embodiment, the culture medium for inducing macrophage culture in step (1) comprises: DMEM complete medium containing 20 ng / ml M-CSF.

[0014] In another preferred embodiment, the digestive solution comprises a neutral protease (Dispase), deoxyribonuclease I (DNase1) Dispase, collagenase I, and the remainder PBS, wherein the volume ratio of the neutral protease (Dispase), deoxyribonuclease I (DNase1), and collagenase I is 1:1~2:0.1~0.2.

[0015] In another preferred embodiment, the tumor cells are mixed with the matrix gel at a ratio of 40,000 to 50,000 cells per 25 μL of matrix gel.

[0016] In another preferred embodiment, the macrophages are selected from the group consisting of: unpolarized M0 macrophages, polarized M1 macrophages, and unpolarized M0 macrophages.

[0017] Macrophages or M2 macrophages; M1 macrophages are preferred.

[0018] In another preferred embodiment, the macrophage is an M1 type macrophage.

[0019] In another preferred embodiment, the co-culture system described in step (3) further includes a polarizing medium that polarizes the macrophages.

[0020] In another preferred embodiment, the polarization medium includes M1 macrophage polarization medium or M2 macrophage polarization medium.

[0021] In another preferred embodiment, the M1 macrophage polarization medium comprises the tumor organoid culture medium and 20 ng / ml of IFN-γ.

[0022] In another preferred embodiment, the M2 macrophage polarization medium comprises the tumor organoid culture medium and 20 ng / ml IL-4.

[0023] In another preferred embodiment, the method further includes the following steps:

[0024] (1) Peripheral blood mononuclear cells were isolated from the peripheral blood of tumor patients. After CD3 positive cells were sorted by magnetic beads, CD3 negative cells were collected. After centrifugation, CD3 negative cells were resuspended in DMEM complete medium and cultured for 24-48 hours. Then, macrophage induction medium was replaced and cultured for 7-8 days. The medium was replaced every 2-3 days to obtain macrophages.

[0025] (2) The tumor tissue from the cancer patient was digested with digestion solution at 37°C and 180-220 rpm for 20-60 minutes. Then the digested tumor cells were passed through a 100 μm cell sieve, and the tumor cells were collected by centrifugation and mixed evenly with matrix gel. Organoid culture medium was added for organoid culture and passage culture. The size of the tumor organoids was then screened using 37 μm and 100 μm cell sieves to obtain humanized tumor organoids of 37 μm-100 μm.

[0026] (3) The well plate was pre-coated with an anti-adhesion reagent, and then the humanized tumor organoids obtained in step (2) were plated and counted. They were then mixed with the macrophages obtained in step (1) at an effector-to-target ratio of 1 to 10:1 to form a co-culture system. The system was then placed in an incubator for culture to obtain the co-culture model of immune cells and tumor organoids used to evaluate anti-tumor activity.

[0027] In another preferred embodiment, the co-culture system in step (3) further includes an inducer to induce the macrophages into M1 macrophages; the inducer is 20 ng / ml IFN-γ, and the induction time is 14 to 20 days.

[0028] In another preferred embodiment, the co-culture system described in step (3) further includes an inducer that induces the macrophages to become M2 macrophages; the inducer is 20 ng / ml IL-4, and the induction time is 14 to 20 days.

[0029] In another preferred embodiment, the target-effect ratio in step (3) is 1:1 or 3:1, preferably 3:1.

[0030] In another preferred embodiment, the target-effect ratio in step (3) is 3:1.

[0031] In another preferred embodiment, the DMEM complete culture medium described in step (1) has the following formulation: DMEM high glucose culture medium, 10% fetal bovine serum (v / v), 1% GlutaMAX (v / v) and 1% penicillin-streptomycin (v / v).

[0032] The digestion solution used in step (2) includes: 10% Dispase, 10% DNase1, 1%~2% collagenase I, and the remainder PBS;

[0033] The organoid culture medium formulation described in step (2) is as follows: Advanced DMEM / F12 medium, B27 additive 1×, Glutamax additive 1×, penicillin and streptomycin 1×, 1.25mM N-acetyl-L-cysteine, 5-10μM SB431542, 1-10μM CHIR99021, 1-10μM BIRB796, 1-10ng / ml human FGF10, and 1-50ng / ml human EGF.

[0034] In another preferred embodiment, the matrix adhesive mentioned in step (2) is Matrigel matrix adhesive.

[0035] In another preferred embodiment, the culture is carried out in an incubator at 37°C and 5% CO2.

[0036] In a second aspect of the invention, a macrophage for evaluating antitumor activity as described in the first aspect of the invention is provided.

[0037] The method for constructing a co-culture model of macrophages and tumor organoids yielded a co-culture model of macrophages and tumor organoids.

[0038] In a third aspect of the present invention, a method for evaluating the antitumor activity of immune cells in vitro is provided, comprising steps (1) to (3) as described in the first aspect of the present invention, and at least one of the following steps:

[0039] (4) Add tracer dyes and fluorescent dyes for detecting tumor cell death to the co-culture system in step (3), and then take continuous pictures of the co-culture system under bright field and fluorescence channel to continuously detect the degree of tumor cell death, the interaction between macrophages and tumor organoids, and the degree of inhibition of tumor organoid growth by macrophages in the co-culture system.

[0040] (5) Add a fluorescent dye for detecting tumor cell death to the co-culture system in step (3), and then use a multi-functional microplate reader to detect the relative fluorescence value of the co-culture system, and calculate and analyze the degree of inhibition of tumor organoid growth by macrophages.

[0041] (6) Detect the concentration levels of effector factors related to cell inflammation in the supernatant of the co-culture system in step (3) to comprehensively evaluate the degree of inhibition of tumor organoid growth by macrophages.

[0042] In another preferred embodiment, the tracer dye in step (4) is Celltracker red CMTPX dye;

[0043] The fluorescent dye mentioned in steps (4) and (5) is Promega Green dye;

[0044] The detection described in step (6) is performed using the CBA multifactor kit;

[0045] The effector factors mentioned in step (6) include IL-1, IL-6, TNF-α and TGF-β, etc.

[0046] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0047] Compared with the prior art, the present invention has the following technical effects:

[0048] (1) This invention provides a breakthrough cell source and induction technology:

[0049] Peripheral blood mononuclear cells can be efficiently induced to differentiate into macrophages in vitro. The technique is stable and reliable, and a high-purity macrophage population can be obtained.

[0050] It significantly reduces the reliance on immune cells derived from tumor tissue, which are difficult to obtain, and makes sample acquisition more convenient (requiring only peripheral blood), especially suitable for situations where clinical samples are limited or fresh tumor tissue cannot be obtained;

[0051] It supports the use of patients' own cells to build models, laying the foundation for personalized research; allogeneic cells can also be used to improve the flexibility and applicability of the models.

[0052] (2) The model provided by the present invention has high biomimicry: the three-dimensional structure combined with autologous / allogeneic peripheral blood-derived immune cells (macrophages) significantly improves the simulation fidelity of human lung cancer TME, especially the immune microenvironment mediated by macrophages.

[0053] (3) Dynamic regulation of macrophage polarization state (M1 / M2 type) in co-cultured organoids: The polarization state and dynamic transformation of macrophages in the tumor microenvironment can be regulated by adding exogenous inducers (IFN-γ, IL-4, etc.).

[0054] (4) The model provided by this invention can be used as a research platform for tumor molecular mechanisms: it provides a powerful tool for in-depth exploration of the molecular mechanisms of the bidirectional interaction between tumor and macrophage in lung cancer progression, immune escape, and treatment resistance.

[0055] (5) The model provided by this invention can be used for drug screening and evaluation: it is suitable for high-throughput screening of immunotherapeutic drugs that target tumor cells, macrophages or their interaction pathways (such as immune checkpoint inhibitors, TAM reprogramming / targeting drugs, cytokine antagonists, etc.), and can evaluate the effect of drugs on macrophage polarization.

[0056] (6) The present invention is relatively simple to operate, has a shorter cycle and controllable cost: compared with organoid models co-cultured with immune cells derived from tumor tissue or pluripotent stem cells, it is easier to obtain samples (peripheral blood + tumor tissue / cells), has a shorter construction cycle, and higher reproducibility and throughput.

[0057] (7) Advantages of peripheral blood-derived macrophages (PBDM) used in this invention in tumor co-culture organoid models compared to iPSC-Mac:

[0058] (7.1) Short cultivation cycle and high efficiency:

[0059] Rapid acquisition of functional cells: PBDM directly isolates terminally differentiated monocytes from the peripheral blood of donors (healthy individuals or patients), and they can differentiate into functionally mature macrophages with only 5-7 days of M-CSF / GM-CSF stimulation. This significantly shortens the experimental cycle and accelerates the establishment and research process of organoid co-culture models.

[0060] Avoiding the risks of long-term culture: Compared with the long multi-stage differentiation process of iPSC-Mac (14-30 days), the short cycle of PBDM significantly reduces the risk of contamination (such as microbial contamination, mycoplasma contamination, etc.) caused by long-term in vitro culture, and improves the stability and reliability of experiments.

[0061] (7.2) Mature cell source with strong functional activity:

[0062] High maturity and metabolic activity: Peripheral blood-derived cells (peripheral blood mononuclear cells) are already in a terminal differentiation state. After short-term stimulation, PBDMs have high functional maturity and active metabolism, and can respond to signals in the organoid microenvironment more quickly and realistically, and participate in immune interactions.

[0063] Preservation of natural functional characteristics: This direct-source approach preserves the natural biological behavior and effector functions of macrophages to the greatest extent, providing immune components that are closer to the physiological state in vivo for co-cultured organoids.

[0064] (7.3) Perfectly reflects donor heterogeneity (the core advantage of individualized modeling):

[0065] Preservation of donor specificity: PBDM is derived directly from the peripheral blood of a specific individual (healthy person or patient), and its genotype, epigenetic characteristics, and response pattern to stimuli faithfully reflect the donor's immune status.

[0066] Constructing personalized / disease-specific models: This feature enables organoids co-cultured with PBDM to accurately preserve and reflect the donor's specific physiological or pathological state (such as the immune characteristics of specific diseases, differences in drug response, and the influence of genetic background). This is an irreplaceable core advantage for conducting personalized treatment research, developing precision medicine strategies, and establishing disease models that truly reflect the heterogeneity of patient populations (such as tumor immune microenvironment models, infection immune models, and inflammatory disease models).

[0067] Reproducing individual immune responses: In drug screening or immunotherapy studies, organoids co-cultured with PBDM can more realistically simulate the unique immune responses of different patients to treatment, which is difficult to achieve with phenotypically homogeneous iPSC-Mac.

[0068] (7.4) The experimental procedure is relatively simplified:

[0069] Compared to the iPSC-Mac process, which requires complex multi-stage differentiation and strict quality control, the PBDM preparation process (isolation and stimulation) is relatively more mature, standardized, and easy to operate, thus lowering the technical threshold. Attached Figure Description

[0070] Figure 1 The image shows the results of immunofluorescence staining of macrophages induced by peripheral blood in this embodiment of the invention. DAPI staining was performed on the cell nuclei, and CD68, a universal marker for macrophages, was detected. 20X represents 20x magnification.

[0071] Figure 2 The results shown are PCR detection results of peripheral blood-induced macrophages in this embodiment of the invention. Among them, CD68 is a universal macrophage marker, CD80 and CD86 are characteristic markers of M1 macrophages, and CD163 is a characteristic marker of M2 macrophages.

[0072] Figure 3 The images shown are microscopic images of tumor organoids co-cultured with macrophages in different proportions according to embodiments of the present invention. The upper image is magnified by 4x; the lower image is magnified by 10x; O+M 3:1 represents tumor cell:macrophage = 3:1; O+M 1:1 represents tumor cell:macrophage = 1:1; O+M 1:3 represents tumor cell:macrophage = 1:3.

[0073] Figure 4 The figures shown are PCR detection results after adding different macrophage polarization inducers to co-cultured tumor organoids in this embodiment of the invention. Among them, CD68 is a universal macrophage marker, CD80 and CD86 are characteristic markers of M1 macrophages, and CD163 is a characteristic marker of M2 macrophages.

[0074] Figure 5 The images shown are microscopic images of co-cultured tumor organoids with different macrophage polarization inducers added in embodiments of the present invention. The upper image is magnified by 4x; the lower image is magnified by 10x; O+M represents the tumor + macrophage control group; O+M+IFN-γ represents the IFN-γ-induced M1 macrophage polarization model; and O+M+IL-4 represents the IL-4-induced M2 macrophage polarization model. Detailed Implementation

[0075] To make the objectives, technical solutions, beneficial effects, and significant advancements of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, all the described embodiments are only some embodiments of the present invention, and not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0076] the term

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0078] As used herein, “including” or “containing” includes “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0079] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents involved in this invention are commercially available.

[0080] Example 1: Extraction of peripheral blood mononuclear cells (PBMCs)

[0081] 1. Centrifugation: Take 5-10 ml of fresh EDTA-anticoagulated whole blood from a healthy person, place it in a centrifuge tube, and centrifuge at 2500 rpm for 10 minutes at room temperature.

[0082] 2. Diluting the sample: After centrifugation, carefully discard the upper plasma layer, and then dilute the remaining sample with sterile PBS at a ratio of 1:1.

[0083] 3. Add Ficoll separation buffer: Add an equal volume of Ficoll separation buffer to a 15ml sterile centrifuge tube, equal to the volume of the diluted blood sample. If the volume of the diluted sample is less than 3ml, add 3ml of Ficoll separation buffer directly.

[0084] 4. Layering: Slowly add the diluted blood sample along the centrifuge tube wall to the surface of the Ficoll separation solution to ensure clear layering and avoid mixing.

[0085] 5. Centrifuge again: Place the centrifuge tube in a centrifuge and centrifuge at 2000 rpm. The centrifugation temperature is room temperature, and the centrifugation time is 25 minutes. Set both acceleration and deceleration to slow to ensure the cell separation effect.

[0086] 6. Aspirate the PBMC loop: After centrifugation, carefully remove the centrifuge tube, avoiding shaking as much as possible. Carefully aspirate the middle white turbid layer, i.e., the PBMC loop, using a pipette.

[0087] 7. Washing PBMCs: Transfer the aspirated PBMC layer to a new sterile 15ml centrifuge tube, then add sterile PBS to a final volume of 10ml and mix gently. Centrifuge at 1500rpm for 6 minutes at room temperature. After centrifugation, aspirate the supernatant, add another 10ml of sterile PBS, mix well, and repeat the centrifugation process once more.

[0088] 8. Red blood cell lysis: After aspirating the supernatant, add 2 ml of red blood cell lysis buffer, mix gently, and incubate at room temperature for 10 min to lyse any remaining red blood cells.

[0089] 9. Centrifuge and discard supernatant: Centrifuge at 1500 rpm for 6 min and discard supernatant.

[0090] 10. Washing the precipitate: Add an appropriate amount of PBS to wash the precipitate, centrifuge at 1500 rpm at room temperature for 6 min, and then remove the supernatant.

[0091] Example 2: Monocyte sorting and macrophage-induced polarization

[0092] 2.1. Incubation of magnetic beads and samples:

[0093] Cell resuspension: Resuspend the cell pellet obtained in step 10 above with 80 μL of buffer.

[0094] Add magnetic beads: Add 20ul CD3 microbeads and mix gently.

[0095] Incubation: Place the mixture in a refrigerator at 2-4℃ and incubate in the dark for 15 minutes to allow the magnetic beads to fully bind to the CD3 antigen on the surface of T cells.

[0096] Washing cells: After incubation, add an appropriate amount of buffer to wash the cells (1-2 ml) to remove unbound magnetic beads.

[0097] Centrifuge and discard supernatant: 300g, 4℃, centrifuge for 10min, then discard supernatant.

[0098] Resuspend: Add 1ml of buffer (Buffer formula: 200ml PBS + 1g BSA + 1.25ml 0.5mMEDTA) to resuspend the cells in preparation for subsequent sorting.

[0099] 2.2. Sorting CD3-positive cells using a column:

[0100] Install the sorting column: Install the sorting column onto the sorter, ensuring it is securely installed.

[0101] Rinse the sorting column: Rinse the sorting column twice with 500ul buffer.

[0102] Cell sorting: After the buffer has drained, add the resuspended cells obtained in step 2.1, and then wash the column 2-3 times with 3 ml of buffer to ensure that the CD3 positive cells can be fully bound to the sorting column.

[0103] 2.3. Collection of CD3 negatively selected cells:

[0104] Collect the CD3 negatively selected cells eluted in step 2.2, 300g, centrifuge at 4℃ for 10min, and discard the supernatant.

[0105] 2.4. Cell resuspension and culture:

[0106] After resuspending the cell pellet from step 2.3 in DMEM complete medium (including DMEM high glucose medium, 10% FBS, 1x GlutaMAX and 1x P / S), transfer the cells to cell culture dishes.

[0107] 2.5. Monocyte adhesion:

[0108] Incubate at 37°C and 5% CO2 for 24-48 hours. Once the mononuclear cells have adhered and grown, replace the culture medium with fresh medium to remove suspended cells.

[0109] 2.6 Macrophage induction: The culture medium used in step 2.5 was DMEM complete medium (i.e. macrophage induction medium) containing 20 ng / ml M-CSF. The cells were cultured at 37°C in a 5% CO2 incubator for 7 days. The culture medium was changed every 2-3 days to induce monocytes to differentiate into macrophages (M0 type).

[0110] 2.7 Cell phenotype verification

[0111] (1) Human monocytes obtained from peripheral blood were induced to differentiate into macrophages in vitro, and their phenotypic characteristics were identified by immunofluorescence staining. CD68 was detected as a universal marker for macrophages.

[0112] Staining results showed that: after DAPI staining, the cell nuclei exhibited blue fluorescence; after CD68-specific staining, positive signals showed green fluorescence. Figure 1 The detection results showed that the macrophages obtained by in vitro induced differentiation highly expressed the CD68 marker.

[0113] (2) To clarify the specific subtypes of the macrophages obtained by in vitro induced differentiation of peripheral blood mononuclear cells, the gene expression levels of subtype-related markers were detected by reverse transcription polymerase chain reaction (RT-PCR). The markers detected included: the universal macrophage marker CD68, the characteristic markers of M1 macrophages CD80 and CD86, and the characteristic marker of M2 macrophages CD163.

[0114] The results showed that the expression level of the CD68 gene was significantly increased in the macrophages obtained by in vitro induced differentiation; while no significant expression of the CD80, CD86, and CD163 genes was detected. Figure 2 Based on this, it was determined that the macrophages were unpolarized M0 type macrophages.

[0115] Example 3: Tumor organoid culture

[0116] 3.1. Digestion of tumor tissue:

[0117] The obtained tumor tissue (taking lung cancer tissue as an example) was cut into small pieces with scissors, and then digested into cell clusters of 3-5 cells using a digestion solution (1ml Dispase + 1ml DNase I + 100ul collagenase I, then supplemented with PBS to 10ml). Care should be taken to control the temperature and time during digestion to ensure cell viability.

[0118] 3.2. Centrifugation to collect the precipitate: Place the digested cell suspension in an environment of 4°C and centrifuge at a speed of 450xg for 10 min, then collect the precipitate.

[0119] 3.3. Washing the precipitate: Resuspend the precipitate in 10 ml of PBS, mix well by pipetting, place it in a 4°C environment again, centrifuge at 450 g for 5 min, discard the supernatant, and take the precipitate.

[0120] 3.4. Red blood cell lysis: Add 10 ml of red blood cell lysis buffer, mix by pipetting, and let stand at room temperature for 10 min to lyse the remaining red blood cells.

[0121] 3.5. Centrifuge again: Centrifuge at 450xg for 5 min at 4℃, discard the supernatant and take the precipitate.

[0122] 3.6. Washing the precipitate: After resuspending with PBS, centrifuge at 450xg for 5 min at 4℃, discard the supernatant, and take the precipitate.

[0123] 3.7. Cell counting: After resuspending the pellet in 1 ml of lung cancer organoid culture medium, cell counting was performed to determine the cell number.

[0124] 3.8. Matrix gel plating: Centrifuge to collect the precipitate, resuspend the precipitate with an appropriate amount of matrix gel (approximately 50,000 cells per 25 μL matrix gel), and plate the cells in droplets of 25 μL matrix gel per well. Then, culture the cells using lung cancer organoid culture medium for 2-3 weeks, changing the medium every 3-4 days.

[0125] The lung cancer organoid culture medium formula is as follows: Advanced DMEM / F12 basal medium, 1x B27, 1x antibiotic-antifungal, 1x Glutamax, 1.25mM N-acetyl-L-cysteine, 5-10μM SB431542, 1-10μM CHIR99021, 1-10μM BIRB796, 1-10ng / ml human FGF10, and 1-50ng / ml human EGF.

[0126] Example 4: Co-culture of tumor organoids and macrophages

[0127] 4.1. Organoid dissociation and counting: The tumor organoids in step 3.8 of Example 3 above are dissociated into single cells, counted, and set aside for later use.

[0128] 4.2. Macrophage collection and counting: Digest the macrophages in step 2.6 of Example 2 above with 3-5 ml TrypLE Express enzyme for 15-30 minutes, collect the cells and count them for later use.

[0129] 4.3. Cell mixing: The dissociated tumor organoid cells and macrophages were mixed at ratios of 3:1, 1:1 and 1:3, respectively, to conduct co-culture system optimization experiments and finally select the most suitable cell culture ratio; macrophage-organoid suspension was obtained, and macrophage-organoid precipitate was obtained by centrifugation.

[0130] The co-culture system optimization experiment included screening the ratio of tumor cells to macrophages, and the process is as follows:

[0131] To determine the optimal co-culture ratio of tumor cells to macrophages for promoting lung cancer organoid growth, an in vitro co-culture system was established. The tumor cells and macrophages were co-cultured at the following cell number ratio:

[0132] Tumor cells:macrophages = 3:1

[0133] Tumor cells:macrophages = 1:1

[0134] Tumor cells:macrophages = 1:3

[0135] The growth of lung cancer organoids in each co-culture group was analyzed and evaluated.

[0136] The results showed that in the co-culture group with a ratio of 1:3 (tumor cells: macrophages), lung cancer organoids exhibited the most significant growth-promoting effect in both size and number. Figure 3 Therefore, this application uses a ratio of tumor cells:macrophages = 1:3 for organoid model construction.

[0137] 4.4. Matrix gel co-culture: Resuspend the macrophage-organoid pellet in an appropriate amount of matrix gel, seeding 40,000 cells per 25 μL of matrix gel, and seeding 25 μL of matrix gel per well. Culture in lung cancer organoid culture medium for 2 weeks, changing the medium every 3-4 days to provide a suitable growth environment for the co-cultured cells.

[0138] Example 5: Co-culture of tumor organoids and polarized macrophages

[0139] 5.1. The macrophages and lung cancer co-cultured organoids in step 4.4 of Example 4 were cultured in M1 polarization medium and M2 polarization medium, respectively. The medium was changed every 3-4 days and cultured for 2 weeks to regulate the polarization state of macrophages in the tumor microenvironment.

[0140] The formulations for each culture medium are as follows:

[0141] M1 polarization medium formulation: lung cancer organoid culture medium + 20 ng / ml IFN-γ.

[0142] M2 polarization medium formulation: lung cancer organoid culture medium + 20 ng / ml IL-4.

[0143] 5.2 Validation of macrophage polarization state in co-culture system:

[0144] According to step 5.1, organoids co-cultured with tumor cells and macrophages at a ratio of 1:3 were treated with inducers IFN-γ and IL-4 for 14 days, and the expression levels of macrophage polarization markers were detected by RT-qPCR.

[0145] The results showed that the expression levels of M1 macrophage markers CD86 and CD80 were increased in the IFN-γ treatment group, while the expression level of M2 macrophage marker CD163 was increased in the IL-4 treatment group. Figure 4 The results confirm that in the co-culture system, the addition of IFN-γ can effectively induce macrophages to M1 polarization, while the addition of IL-4 can effectively induce macrophages to M2 polarization.

[0146] 5.3 Effects of macrophages with different polarization on tumor organoid growth

[0147] Based on the macrophage polarization model validated in step 5.3 (IFN-γ-induced M1 type, IL-4-induced M2 type), the effects of different polarized macrophages on tumor organoid growth were observed under a microscope.

[0148] The results showed that, compared with the control group (without inducing agent treatment), the number of tumor organoids was significantly reduced in the IFN-γ treatment group, while the number of tumor organoids was slightly increased in the IL-4 treatment group. This confirms that the polarization state of macrophages plays a regulatory role in the growth behavior of tumor organoids.

[0149] The preferred embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and the devices and structures not described in detail should be understood as being implemented in a conventional manner in the art. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the scope of the present invention. This does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the present invention's technical solutions still fall within the protection scope of the present invention.

Claims

1. A method for constructing a macrophage and tumor organoid co-culture model for evaluating antitumor activity, characterized in that, Includes the following steps: (1) Peripheral blood mononuclear cells were isolated from peripheral blood of healthy individuals, and macrophage cells were induced to be obtained after macrophage pretreatment culture and induction culture. (2) The tumor tissue from the cancer patient was digested with digestive fluid, the digested tumor cells were collected by centrifugation and mixed evenly with matrix gel, and then added to tumor organoid culture medium for tumor organoid culture and passage culture to obtain humanized tumor organoids. (3) The humanized tumor organoids obtained in step (2) and the macrophages obtained in step (1) are mixed at an effector-target ratio of 1 to 10:1 to form a co-culture system, and then placed in an incubator for culture to obtain the macrophage and tumor organoid co-culture model used to evaluate anti-tumor activity.

2. The construction method according to claim 1, characterized in that, The tumor is lung cancer.

3. The construction method according to claim 1, characterized in that, The macrophages are selected from the following group: unpolarized M0 macrophages, polarized M1 macrophages or M2 macrophages; preferably M1 macrophages.

4. The construction method as described in claim 1, characterized in that, The method further includes the following steps: (1) Peripheral blood mononuclear cells were isolated from peripheral blood of healthy individuals. After CD3 positive cells were sorted by magnetic beads, CD3 negative cells were collected. After centrifugation, CD3 negative cells were resuspended in DMEM complete medium and cultured for 24-48 hours. Then, macrophage induction medium was replaced and cultured for 7-8 days. The medium was replaced every 2-3 days to obtain macrophages. (2) The tumor tissue from the cancer patient was digested with digestion solution at 37°C and 180-220 rpm for 20-60 minutes. Then the digested tumor cells were passed through a 100 μm cell sieve, and the tumor cells were collected by centrifugation and mixed evenly with matrix gel. Organoid culture medium was added for organoid culture and passage culture. The size of the tumor organoids was then screened using 37 μm and 100 μm cell sieves to obtain humanized tumor organoids of 37 μm-100 μm. (3) The well plate was pre-coated with an anti-adhesion reagent, and then the humanized tumor organoids obtained in step (2) were plated and counted. They were then mixed with the macrophages obtained in step (1) at an effector-to-target ratio of 1 to 10:1 to form a co-culture system. The system was then placed in an incubator for culture to obtain the co-culture model of immune cells and tumor organoids used to evaluate anti-tumor activity.

5. The construction method according to claim 1, characterized in that, The co-culture system described in step (3) also includes an inducer that induces the macrophages to become M1 macrophages; the inducer is 20 ng / ml of IFN-γ, and the induction time is 14 to 20 days.

6. The construction method according to claim 1, characterized in that, The effective-to-target ratio in step (3) is 1:1 or 3:1, preferably 3:

1.

7. The construction method according to claim 1, characterized in that, The DMEM complete culture medium mentioned in step (1) has the following formula: DMEM high glucose medium, 10% fetal bovine serum (v / v), 1% GlutaMAX (v / v) and 1% penicillin-streptomycin (v / v). The digestion solution used in step (2) includes: 10% by volume neutral protease (Dispase), 10% by volume deoxyribonuclease I (DNase1), 1% to 2% by volume collagenase I, and the remainder PBS; The organoid culture medium formulation described in step (2) is as follows: Advanced DMEM / F12 medium, B27 additive 1×, Glutamax additive 1×, penicillin and streptomycin 1×, 1.25mM N-acetyl-L-cysteine, 5-10μM SB431542, 1-10μM CHIR99021, 1-10μM BIRB796, 1-10ng / ml human FGF10, and 1-50ng / ml human EGF.

8. The macrophage and tumor organoid co-culture model constructed according to the method for constructing a macrophage and tumor organoid co-culture model for evaluating antitumor activity as described in claim 1.

9. A method for evaluating the antitumor activity of immune cells in vitro, characterized in that: Includes steps (1) to (3) as described in claims 1, and at least one of the following steps: (4) Add tracer dyes and fluorescent dyes for detecting tumor cell death to the co-culture system in step (3), and then take continuous pictures of the co-culture system under bright field and fluorescence channel to continuously detect the degree of tumor cell death, the interaction between macrophages and tumor organoids, and the degree of inhibition of tumor organoid growth by macrophages in the co-culture system. (5) Add a fluorescent dye for detecting tumor cell death to the co-culture system in step (3), and then use a multi-functional microplate reader to detect the relative fluorescence value of the co-culture system, and calculate and analyze the degree of inhibition of tumor organoid growth by macrophages. (6) Detect the concentration levels of effector factors related to cell inflammation in the supernatant of the co-culture system in step (3) to comprehensively evaluate the degree of inhibition of tumor organoid growth by macrophages.

10. The method according to claim 9, characterized in that, The tracer dye mentioned in step (4) is Celltracker red CMTPX dye; The fluorescent dye mentioned in steps (4) and (5) is Promega Green dye; The detection described in step (6) is performed using the CBA multifactor kit; The effector factors mentioned in step (6) include IL-1, IL-6, TNF-α and TGF-β.

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